Topical antiviral formulations

ABSTRACT

The disclosure provides novel topical antiviral pharmaceutical compositions comprising one or more antiviral compounds and 2-deoxy-D-glucose in the form of lip-balms, creams and ointments. A specific embodiment discloses a lip-balm composition comprising acyclovir and 2-deoxy-D-glucose.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/102,729, filed Oct. 3, 2008, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

The disclosure provides novel topical antiviral pharmaceuticalcompositions comprising an antiviral compound such as acyclovir and2-deoxy-D-glucose.

2. Description of the Related Art

Herpes simplex virus (HSV) infections are ubiquitous, with approximately80% of the adult population infected with HSV type 1 and approximately20% of the adult population also infected with HSV type 2. HSV type 1 isthe cause of herpes labialis, also called orofacial herpes, or coldsores, and HSV encephalitis. HSV type 2 is the primary cause of initialand recurrent genital herpes, and neonatal HSV. The typicalmanifestation of a primary HSV-1 or HSV-2 genital infection is clustersof inflamed papules and vesicles on the outer surface of the genitalsresembling cold sores. (Gupta et al., 2007, “Genital herpes” Lancet 370(9605): 2127-37).

Herpes viruses cycle between periods of active disease—presenting asblisters containing infectious virus particles—that last 2-21 days,followed by a remission period, during which the sores disappear. ManyHSV infected people experience recurrence within the first year ofinfection. During recurrence fewer lesions are likely to develop,lesions are less painful, and lesions heal faster, than those occurringduring the primary infection. Subsequent outbreaks tend to be periodicor episodic, occurring on average four to five times a year when notusing antiviral therapy.

Treatment of initial HSV infection, and reactivated latent HSVinfection, typically includes topical application of an antiviralnucleoside composition to lesions at outbreak. Current prescriptiontopical treatments for herpes labialis include Zovirax® cream (5%acyclovir, GlaxoSmithKline/Biovail Pharmaceuticals, Inc.) which is FDAapproved for the treatment of recurrent herpes labialis (cold sores) inadults and adolescents (12 years of age and older). Another topicaltreatment is Denavir® (1% penciclovir, Novartis), which is FDA approvedfor the treatment of recurrent herpes labialis (cold sores) in adults.These treatments are noted to inhibit viral replication; shorten healingtime and duration of symptoms; and are soothing. Acyclovir andpenciclovir are antiviral nucleosides. Zovirax® is applied five times aday for four days. Denavir® is applied every two hours during the dayfor four days.

Zovirax® ointment (5% acyclovir, GlaxoSmithKline/BiovailPharmaceuticals, Inc.) is approved for topical administration and isindicated in the management of initial genital herpes and in limitednon-life-threatening mucocutaneous Herpes simplex virus infections inimmunocompromised patients. Side effects include mild pain uponapplication, pruritis, and rash.

Several patents list acyclovir as an ingredient in topical formulationsfor the treatment of herpes simplex genitalis and herpes labialis.

Jones and White, U.S. Pat. No. 4,963,555 is listed in the electronicorange book for Zovirax® topical cream, 5% acyclovir. The '555 patentdiscloses a topical pharmaceutical formulation for use in treating virusinfections of the skin or mucosa and containing9-(2-hydroxyethoxymethyl)guanine, or a salt or ester thereof, whichcomprises a dispersed oil phase and a continuous aqueous phasecontaining therein water, at least 30 wt % of a polyhydric alcohol andsolublized acyclovir.

Sintov et al., U.S. Pat. No. 5,585,379, disclose antiviral topicalpharmaceutical compositions containing acyclovir dispersed in an aqueousgel carrier containing a gelling agent; a water-soluble carboxylic ordicarboxylic acid salt, such as sodium or potassium oleate; and apolyhydroxy compound such as glycerine, propylene glycol andpolyethylene glycol.

Kaufman and Faro, 1999, Clin. Obstet. Gynecol., 28 (1): 152-163 providea review article describing the clinical features of genital herpesvirus infection as well as a discussion of some of the epidemiologicfeatures which may be related to the increased frequency with which thisdisease is being seen and describing treatment of genital herpesinfections.

Spruance et al., 2002, Antimicrob. Agents Chemother. 46 (7): 2238-2243,provide a description of a placebo-controlled clinical trial of a 5%acyclovir topical cream for the treatment of herpes labialis wherein themean duration of episodes was 4.3 days for patients treated withacyclovir cream and 4.8 days for those treated with the vehicle control.

Certain patents list 2-deoxy-D-glucose as an ingredient in topicalformulations for the treatment of herpes simplex genitalis and herpeslabialis.

Blough, U.S. Pat. No. 4,603,122, issued Jul. 29, 1986, discloses amethod of treating herpes virus infection by the administration of2-deoxy-D-glucose. For example, for herpes labialis, the topicaltreatment consisted of two to three drops of a 50 mM solution of2-deoxy-D-glucose in sterile anhydrous glycerol.

Blough, U.S. Pat. No. 4,315,001, issued Feb. 9, 1982 discloses a methodof treating herpes simplex virus by the administration of2-deoxy-D-glucose.

Blough et al., 1979, J. Am. Med. Assoc, 241(26), 2798-280, describe aclinical study of the treatment of genital herpes infections with2-deoxy-D-glucose.

Patient satisfaction with current remedies, particularly forHSV-1—herpes labialis, has been poor. Unfortunately, topical acyclovirtherapy lacks efficacy as compared to oral or parenteraladministrations. The drug concentration in the skin after localapplication is 2-3 times lower than after given orally (Greg et al.,1992, J. Invest. Dermatol. 98:856-63). It is speculated that the lack ofefficacy of topical treatments may be related to the poorwater-solubility and lipophilicity of acyclovir, resulting in itsinadequate skin or mucous membrane partitioning ability. However, it isstill considered safer to give the drug locally.

It is possible that viral resistance to acyclovir should be consideredin patients who show poor clinical response during therapy. Resistanceof HSV can result from qualitative and quantitative changes in the viralTK and/or DNA polymerase; particularly noted in immunocompromisedpatients. Clearly, there is room for improvement in topical antiviralcompositions for the rapid, efficacious treatment of HSV infections.

It is herein disclosed that a novel topical formulation comprising anantiviral compound and 2-deoxy-D-glucose has been found to be effectivein the treatment of HSV infection.

SUMMARY

The disclosure provides a topical antiviral pharmaceutical compositioncomprising one or more antiviral compounds and 2-deoxy-D-glucose. In oneembodiment, the antiviral compound is selected from one or more ofacyclovir, vidarabine, azidothymidine, ganciclovir, famciclovir,penciclovir, brivudine, cidofovir, trifluridine, and foscarnet; or apharmaceutically acceptable salt or hydrate thereof. In a specificembodiment, the antiviral compound is acyclovir.

In one embodiment, the topical composition is in a form selected from alip-balm, stick, cream or ointment. In one specific aspect, the topicalcomposition is in a lip-balm form. In one aspect, the compositionfurther comprises one or more polyethylene glycols. In another aspect,the composition optionally further comprises one or more sweeteners orflavorings. In one aspect, the sweetener is stevioside. In anotheraspect, the flavoring is spearmint oil.

In one embodiment, the composition comprises from about 3 wt % to about7 wt % of acyclovir and from about 0.1 wt % to about 5 wt % of2-deoxy-D-glucose. In one aspect, the composition comprises about 5 wt %acyclovir and about 0.2 wt % 2-deoxy-D-glucose.

The disclosure also provides a method of treating a herpes viralinfection of the skin or mucosa of a mammal which comprises applying tothe skin or mucosa a topical composition comprising a therapeuticallyeffective amount of an antiviral compound, or a pharmaceuticallyacceptable salt thereof, and 2-deoxy-D-glucose. In one embodiment, themethod of treating comprises applying to the skin or mucosa acomposition comprising one or more antiviral compounds selected from thegroup consisting of acyclovir, vidarabine, azidothymidine, ganciclovir,famciclovir, penciclovir, brivudine, cidofovir, trifluridine, andfoscarnet; and 2-deoxy-D-glucose. In a specific embodiment, the methodof treating comprises applying to the skin or mucosa a compositioncomprising acyclovir and 2-deoxy-D-glucose. In one aspect, the method oftreating involves topical application of the composition in a formselected from a lip-balm, stick, cream or ointment. In one specificaspect, the topical composition is in a lip-balm form. In one aspect,the method of treating involves topical application of the compositionwhich further comprises one or more polyethylene glycols. In anotheraspect, the method of treating involves topical application of thecomposition further comprising one or more sweeteners or flavorings. Inone aspect, the sweetener is stevioside. In another aspect, theflavoring is spearmint oil. In one aspect the method is for treatment oflatent infection of herpes simplex type 1 infection. In another aspect,the method is for treatment is for latent infection of herpes simplextype 2 infection.

In one aspect, the method of treating involves topical application ofthe composition which comprises from about 3 wt % to about 7 wt % ofacyclovir and from about 0.1 wt % to about 5 wt % of 2-deoxy-D-glucose.In one aspect, the composition comprises about 5 wt % of the acyclovirand about 0.2 wt % of 2-deoxy-D-glucose.

The disclosure further provides a method of reducing the duration of aherpes viral infection outbreak of the skin or mucosa of a mammal whichcomprises applying to the skin or mucosa a topical compositioncomprising a therapeutically effective amount of an antiviral compound,or a pharmaceutically acceptable salt thereof, and 2-deoxy-D-glucose. Inone aspect the method is for treatment of latent infection of herpessimplex type 1 infection. In another aspect, the method is for treatmentis for latent infection of herpes simplex type 2 infection.

In one embodiment, the method of treating comprises applying to the skinor mucosa a composition comprising one or more antiviral compoundsselected from the group consisting of acyclovir, vidarabine,azidothymidine, ganciclovir, famciclovir, penciclovir, brivudine,cidofovir, trifluridine, and foscarnet; and 2-deoxy-D-glucose. In aspecific embodiment, the method of treating involves topical applicationof a composition comprising the antiviral compound acyclovir and2-deoxy-D-glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a concentration/response graph for in vitro inhibition ofHSV-1 infection of adult human dermal microvascular endothelial cellswhen treated with acyclovir, 2-deoxy-D-glucose or an equimolarcombination of the two compounds.

FIG. 2 shows a concentration/response graph for in vitro inhibition ofHSV-2 infection of adult human dermal microvascular endothelial cellswhen treated with acyclovir, 2-deoxy-D-glucose or an equimolarcombination of the two compounds.

DETAILED DESCRIPTION

The disclosure provides a novel topical antiviral pharmaceuticalcomposition comprising one or more antiviral compounds and2-deoxy-D-glucose. The disclosure provides topical antiviralcompositions in the form of a lip-balm, stick, cream, ointment, lotion,gel, plaster, or pen. A specific embodiment discloses a lip-balm stickcomposition comprising acyclovir and 2-deoxy-D-glucose.

According to one embodiment, the present invention provides apharmaceutical composition, which comprises a therapeutically effectiveamount of an antiviral compound, or a pharmaceutically acceptable saltor ester thereof, and 2-deoxy-D-glucose, together with apharmaceutically acceptable diluent or carrier.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which may serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21)silica gel; and (22) other non-toxic compatible substances employed inpharmaceutical formulations.

“Optional” or “optionally” means that the subsequently describedingredient may or may not be included in disclosed compositions.“Optionally” is inclusive of embodiments in which the describedingredient is present and embodiments in which the described ingredientis not present.

The use of “wt %” and “w/w” indicates the relative weight percent of aspecified ingredient when compared to the weight of the totalformulation, unless otherwise specified.

Also included in the family of compounds of the present invention arethe pharmaceutically acceptable salts, and esters thereof. The term“pharmaceutically acceptable salts” embraces salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically acceptable. Suitable pharmaceutically acceptable acidaddition salts of compounds of the present invention can be preparedfrom inorganic acid or from an organic acid. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric, and phosphoric acid. Appropriate organic acids may be selectedfrom aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic, stearic,cyclohexylaminosulfonic, algenic, galacturonic acid. Suitablepharmaceutically-acceptable base addition salts of compounds of thepresent invention include metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom N,N′-dibenzylethyleneldiamine, choline, chloroprocaine,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocain. All of these salts may be prepared by conventional means fromthe corresponding compounds of by reacting, for example, the appropriateacid or base with the compounds of the present invention.

As used herein, the term “pharmaceutically acceptable ester” refers toesters which hydrolyze in vivo and include, but are not limited to,those that break down readily in the human body to leave the parentcompound or a salt thereof Suitable ester groups include, for example,those derived from pharmaceutically acceptable aliphatic carboxylicacids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioicacids, in which each alkyl or alkenyl moiety advantageously has not morethan 6 carbon atoms. Examples of particular esters include formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.

The compositions may be formulated for any route of administration, inparticular for oral, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal administration. The compositions may beformulated in any conventional form, for example, as tablets, capsules,caplets, solutions, suspensions, dispersions, syrups, sprays, gels,suppositories, patches and emulsions. In certain specific embodiments,transdermal, topical formulations are disclosed.

The term “inhibit” or “inhibiting” refers to a statistically significantand measurable reduction in activity, preferably a reduction of at leastabout 10% versus control, more preferably a reduction of about 20% ormore, still more preferably a reduction of about 30% or more.

A “therapeutically effective amount” is an amount of a compound of thepresent invention or a combination of two or more such compounds, suchas an antiviral compound and 2-deoxy-D-glucose, which inhibits, totallyor partially, the progression of the condition or alleviates, at leastpartially, one or more symptoms of the condition. A therapeuticallyeffective amount can also be an amount that is prophylacticallyeffective. The amount that is therapeutically effective will depend uponthe patient's size and gender, the condition to be treated, the severityof the condition and the result sought. For a given patient andcondition, a therapeutically effective amount can be determined bymethods known to those of skill in the art. For example, in reference tothe treatment of a latent herpes viral infection using the compositionsof the present invention, a therapeutically effective amount refers tothat amount of an antiviral compound which has the effect of (1)reducing the pain, tingling, burning or itching of the outbreak, (2)reducing the duration of the outbreak, (3) reducing the recurrence ofoutbreaks, and/or, (4) relieving to some extent (or, preferably,eliminating) one or more other symptoms associated with the outbreaksuch as, for example, ulceration, crusting, weeping and scabbing.

The term “subject”, or “patient”, refers to an animal, for example amammal, who is the object of treatment. Preferably, the patient is ahuman. The subject, or patient, may be either male or female.

The term “about” when used to refer to a therapeutically effectiveamount of an antiviral compound, or another compound, includes thespecified amount ±10%.

HSV-1 and 2 are virtually identical, sharing approximately 50% of theirDNA and having over 80% of common antigens. Both types infect the body'smucosal surfaces, usually the mouth or genitals, and then establishlatency in the nervous system. For both types, at least two-thirds ofinfected people have no symptoms, or symptoms too mild to notice.However, both types can recur and spread even when no symptoms arepresent. By the time they are teenagers or young adults, about 50% ofAmericans have HSV-1 antibodies in their blood. By the time they areover age 50, some 80-90% of Americans has HSV-1 antibodies. Bycomparison, almost all HSV-2 is encountered after childhood, when peoplebecome sexually active.

HSVs tend to infect cells of ectodermal origin. After direct exposure toinfectious material (i.e., saliva, genital secretions), initial viralreplication occurs at the entry site in the skin or mucous membrane.After the initial nonspecific inflammatory response to primaryinfection, specific antibody response occurs in a few days, followed bya cellular immune response in the second or third week. In persons withcellular immune defects, primary HSV infection can result inlife-threatening disseminated disease. In rare cases, the initialreplication may lead to disease and life-threatening infection (e.g.,encephalitis). After retrograde axonal flow from neurons at the viralpoint of entry and local replication, the viral genome becomes latent.

HSV latency is defined as the ability to remain in a nonreplicating formin the dorsal root ganglia of the CNS. No viral particles are producedduring latency. A stimulus (e.g., physical or emotional stress, fever,ultraviolet light) reactivates the virus in the form of skin vesicles ormucosal ulcers, with symptoms less severe than those of the primaryinfection. Latent HSV can be reactivated from the trigeminal, sacral,and vagal ganglia. Herpes labialis is the most frequent clinical sign ofreactivation of HSV infection.

Herpes labialis, also called orofacial herpes, or cold sores, is mostoften caused by Herpes Simplex Virus Type 1 (HSV-1). Reactivation ofHSV, predominantly HSV-1, is rarely associated with systemic signs andsymptoms; rather a prodrome of localized pain, tingling (parasthesia),burning, or itching frequently precede recurrent orolabial lesions.

Herpes labialis outbreak proceeds through several stages. Outbreaks canbe triggered by any one of several factors including stress, sunlight,fatigue, fever, illness, poor diet, food allergy, and hormonal changes.The prodrome stage may last from a few hours to a few days and isgenerally accompanied by a tingling or burning sensation around the lipsor nose. The blister stage occurs within a day or two of the prodromestage, there is the first visible sign of clusters of small blisters.The blister stage is followed by the weeping/ulcer stage. This stage ischaracterized by rupture of the blisters leaving a shallow reddishulceration. This is the most painful and contagious stage. Viralshedding occurs generally during the first 4-5 days of outbreakcommencing during the prodrome stage. The weeping ulcer stage isfollowed by the crusting stage. A scab with a brown crust forms. If thescab cracks, the sufferer will experience itching, burning and bleeding.The healing stage follows the crusting stage. If a scab has formed, itwill flake off during the healing stage.

In recurrent orolabial herpetic infection, the lesions tend to recur atthe same site as the original lesions. Pain is most severe at the onsetof infection and diminishes after 4-5 days. Patients with primaryimmunodeficiencies, AIDS, malignancy, malnutrition, or burns andtransplant recipients (e.g., bone marrow, organs) receivingimmunosuppressive therapy can have unusually severe HSV infections.Beginning antiviral treatment when prodrome is experienced can reducethe appearance and duration of lesions in some individuals.

Genital herpes (herpes genitalis) is a sexually transmitted disease(STD) caused by the herpes simplex viruses type 1 (HSV-1) or type 2(HSV-2). Most genital herpes is caused by HSV-2. Most individuals haveno or only minimal signs or symptoms from HSV-1 or HSV-2 infection. Whensigns do occur, they typically appear as one or more blisters on oraround the genitals or rectum. The blisters break, leaving tender ulcers(sores) that may take two to four weeks to heal the first time theyoccur. Typically, another outbreak can appear weeks or months after thefirst, but it almost always is less severe and shorter than the firstoutbreak. Although the infection can stay in the body indefinitely, thenumber of outbreaks tends to decrease over a period of years. As inherpes labialis, beginning antiviral treatment when prodrome isexperienced can reduce the appearance and duration of lesions.

The disclosure provides topical antiviral compositions which reduce boththe severity of symptoms and the duration of HSV infection outbreak. Thenovel antiviral compositions comprise one or more antiviral compoundsand 2-deoxy-D-glucose for the treatment of HSV type 1 and HSV type 2infections. In one aspect, the disclosure provides a method for reducingreactivation of a latent infection of herpes viruses in a humancomprising topically administering a pharmaceutical compositioncomprising a therapeutically effective amount of an antiviral compoundand 2-deoxy-D-glucose.

Antiviral compounds to be combined with 2-deoxy-D-glucose in thecompositions of the present disclosure are selected from one or more of,but are not limited to, certain antiviral nucleosides includingAcyclovir (9-(2-hydroxyethoxymethyl)guanine;2-Amino-1,9-dihydro-9-((2-hydroxyethoxy)methyl)-6H-purin-6-one; CAS No.59277-89-3; M.W. 225.207 g/mol), Acyclovir sodium(2-Amino-1,9-dihydro-9-((2-hydroxyethoxy)methyl)-6H-purin-6-onemonosodium salt, CAS No. 69657-51-8; M.W. 247.189 g/mol), Vidarabine(adenine arabinoside; CAS No. 5536-17-4), Vidaribine monohydrate(9-beta-D-Arabinofuranosyl-9H-purine-6-amine monohydrate; CAS No.24356-66-9); Azidothymidine (AZT, Retrovir, Zidovudine,3-azido-3″-deoxythymidine; CAS No. 30516-87-1), Ganciclovir(9-(1,3-dihydroxy-2-propoxy)methylguanine, DHPG; CAS No. 82410-32-0),Ganciclovir sodium(9-((2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl)guanine, Sodium;2-amino-1,9-dihydro-9-((2-hydroxy-1-(hydroxymethyl)ethoxy)methyl)-6H-purin-6-onemonosodium salt; CAS No. 107910-75-8 or 84245-13-6); Famciclovir(1,3-Propanediol, 2-(2-(2-amino-9H-purin-9-yl)ethyl)-, diacetate(ester); CAS No. 104227-87-4), Penciclovir(9-(4-Hydroxy-3-(hydroxymethyl)butyl)guanine; CAS No. 39809-25-1),Brivudine ((E)-5-(2-Bromovinyl)-2′-deoxyuridine; CAS No. 69304-47-8);Cidofovir (Phosphonic acid,((2-(4-amino-2-oxo-1(2H)-pyrimidinyl)-1-(hydroxymethyl)ethoxy)methyl)-,(S)-; CAS No. 113852-37-2); Trifluridine (5-Trifluorothymidine; CAS No.70-00-8) and any salts or hydrates thereof. The antiviral nucleosidesmay be obtained commercially, or produced by synthetic procedures knownin the art, for example, as described in Izawa and Shiragami, 1998,“Practical syntheses of antiviral nucleosides”, Pure & Appl. Chem.,70(2): 313-318.

In one embodiment, the topical antiviral compositions contain from abouta total of about 0.1 to about 10 wt % of one or more antiviral compoundsand 2-deoxy-D-glucose. In one aspect, the topical antiviral compositioncomprises from about 3 wt % to about 7 wt % of an antiviral compound. Inanother aspect, the topical antiviral compound is present from about 0.5wt % to about 4 wt % of an antiviral compound. In yet anotherembodiment, the topical antiviral compound is present in from about 1%to about 3% weight compared to the total weight of the formulation.

Acyclovir is a synthetic purine nucleoside analogue with in vitro and invivo inhibitory activity against herpes simplex virus types 1 (HSV-1), 2(HSV-2), and varicella-zoster virus (VZV). The inhibitory activity ofacyclovir is highly selective due to its affinity for the enzymethymidine kinase (TK) encoded by HSV and VZV. This viral enzyme convertsacyclovir into acyclovir monophosphate, a nucleotide analogue. Themonophosphate is further converted into diphosphate by cellularguanylate kinase and into triphosphate by a number of cellular enzymes.In vitro, acyclovir triphosphate stops replication of herpes viral DNA.This is accomplished in 3 ways: 1) competitive inhibition of viral DNApolymerase, 2) incorporation into and termination of the growing viralDNA chain, and 3) inactivation of the viral DNA polymerase. The greaterantiviral activity of acyclovir against HSV compared to VZV is due toits more efficient phosphorylation by the viral TK.

It is noted in the Zovirax® (acyclovir) Cream 5% prescribing informationthat resistance of HSV to acyclovir can result from quantitative andqualitative changes in the viral TK and/or DNA polymerase. Clinicalisolates of HSV with reduced susceptibility to acyclovir have beenrecovered from immunocompromised patients, especially with advanced HIVinfection. Most of the acyclovir-resistant mutants isolated thus farfrom immunocompromised patients have been TK-deficient mutants, othermutants involving the viral TK-gene (TK partial and TK altered) and DNApolymerase have been isolated. TK-negative mutants cause severe diseasein immunocompromised patients and infants. The possibility of viralresistance to acyclovir should be considered in patients who show poorclinical response during therapy. A multi-therapeutic approach totopical antiviral compositions is herein disclosed as one solution tothe problem of resistance. The disclosure provides compositionscomprising one or more antiviral compounds and 2-deoxy-D-glucose.

The antiviral action of 2-deoxy-D-glucose has been known for decades.2-deoxy-D-glucose (deoxyglucose; 2-deoxy-D-arabino-hexose;2-deoxy-D-mannose; 2-desoxy-D-glucose; CAS No. 154-17-6; M.W. 164.156g/mol) is incorporated directly into glycoproteins and glycolipids andappears to block the cellular synthesis of the major glycosylatedpolypeptide of the herpes virus. For example, in HSV-infected cellstreated with 2-deoxy-D-glucose, hematosides are notably reduced, with anaccumulation of precursor molecules, namely, the ceramide backbone. Inaddition, 2-deoxy-D-glucose appears to prevent the synthesis andtransport of nonstructural glycolipids. 2-Deoxy-D-glucose, therefore, isa rational and effective chemotherapetic agent in the treatment ofgenital herpes because of its ability to prevent the synthesis ofmacromolecules required for the envelope biogenesis and recognitionphenomenon. 2-deoxy-D-glucose may be purchased commercially. In oneembodiment, the topical antiviral compositions contain from about 0.1 toabout 10 wt % of 2-deoxy-D-glucose. In one aspect, the topical antiviralcomposition comprises from about 0.1% to about 5% of 2-deoxy-D-glucose.

The compositions of the disclosure contain a weight ratio of antiviralcompound to 2-deoxy-D-glucose from about 1:100 to about 100:1. In oneembodiment, the weight ratio of antiviral compound to 2-deoxy-D-glucoseis from about 1:30 to about 30:1. In a specific aspect, the ratio ofacyclovir to 2-deoxy-D-glucose is about 25:1. In one specific aspect,the molar ratio of acyclovir to 2-deoxy-D-glucose is about 18:1. Inanother specific aspect, the molar ratio of acyclovir to2-deoxy-D-glucose is about 1:1.

In a specific embodiment, the antiviral compound is acyclovir. In aspecific aspect, the topical antiviral composition comprises from about3 wt % to about 7 wt % acyclovir. In a preferred specific aspect, thetopical antiviral composition comprises from about 5 wt % acyclovir. Inanother specific aspect, the topical antiviral composition comprisesfrom about 0.1 wt % to about 5 wt % of 2-deoxy-D-glucose.

In another embodiment, the disclosure provides a formulation comprisingpenciclovir and 2-deoxy-D-glucose. Penciclovir is an acyclic nucleosideanalog available as a 1% topical cream for the treatment of recurrentherpes labialis (cold sores) in adults (Denavir®, Novartis). In oneaspect, the penciclovir is present in from about 0.1% to about 5 wt % ofthe weight of the formulation. In a specific aspect, the penciclovir ispresent in about 1 wt % of the total weight of the formulation. Inanother specific aspect, the topical antiviral composition comprisesfrom about 0.1% to about 5 wt % of 2-deoxy-D-glucose.

In another embodiment, the disclosure provides a topical formulationcomprising the non-nucleoside antiviral compound foscarnet and2-deoxy-D-glucose. Foscarnet is a phosphonic acid derivative marketed asfoscarnet sodium in an injectable formulation (Foscavir®, AstraZeneca).Foscarnet is an antiviral medication used to treat herpes viruses,including drug resistant cytomegalovirus (CMV) and herpes simplexviruses types 1 and 2 (HSV-1 and HSV-2). Topical foscarnet has been usedto treat severe genital ulceration due to acyclovir-resistant HSV-2. SeePechere et al., Dermatology, 1998; 197:278-280. In one aspect, thefoscarnet is present in from about 1 wt % to about 5 wt % of the totalweight of the formulation. In a specific aspect, foscarnet is present inabout 2.4 wt % of the weight of the formulation. In another specificaspect, the topical antiviral composition comprises from about 0.1% toabout 5 wt % of 2-deoxy-D-glucose.

In one embodiment, the topical antiviral composition can optionallyfurther comprise one or more sweeteners. The optional sweetener is addedto increase patient acceptability and compliance with the recommendeddosing schedule. However, the sweetener may not be selected from asimple sugar, as the presence of the simple sugar may interfere with theaction of the 2-deoxy-D-glucose. The sweetener may be selected from asynthetic or natural sweetener, for example, aspartame, a cyclamate,saccharin, acesulfame salts, neo-hesperidin dihydrochalcone, sucralose,alitame, astevia, stevioside, talin, glycerrhizin, thaumatin, xylitol,and mixtures thereof. The term saccharin as used herein includessaccharin itself, saccharin acids, and saccharin salts such as sodiumsaccharin. In one aspect, the sweetener is stevioside. The sweetener isoptionally present from about 0.1% to about 5 wt % of the weight of thetopical antiviral compositions.

In another embodiment, the topical antiviral composition can optionallyfurther comprise one or more flavoring agents. The optional flavoringagent is added to increase patient acceptability and compliance with therecommended dosing schedule. The flavoring agents that may be usedinclude those flavors known to the skilled artisan, such as natural andartificial flavors. These flavorings may be chosen from synthetic flavoroils and flavoring aromatics and/or oils, oleoresins and extractsderived from plants, leaves, flowers, fruits, and so forth, andcombinations thereof. Non-limiting representative flavor oils includespearmint oil, cinnamon oil, oil of wintergreen (methyl salicylate),peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, thymeoil, cedar leaf oil, oil of nutmeg, allspice, oil of sage, mace, oil ofbitter almonds, and cassia oil. Also useful flavorings are artificial,natural and synthetic fruit flavors such as vanilla, and citrus oilsincluding, without limitation, lemon, orange, lime, grapefruit, andfruit essences including apple, pear, peach, grape, strawberry,raspberry, cherry, plum, pineapple, apricot and so forth. Theseflavoring agents may be used in liquid or solid form and may be usedindividually or in admixture. Commonly used flavors include mints suchas peppermint, menthol, artificial vanilla, cinnamon derivatives, andvarious fruit flavors, whether employed individually or in admixture.Other useful flavorings include aldehydes and esters such as cinnamylacetate, cinnamaldehyde, citral diethylacetal, dihydrocarvyl acetate,eugenyl formate, p-methylamisol, and so forth may be used. In a specificaspect, the flavoring is spearmint oil. The flavor is optionally presentfrom about 0.1% to about 5% by weight of the topical antiviralcomposition.

In a further embodiment, the topical antiviral composition canoptionally further comprise one or more colorants and/or opacifiers inorder to blend with the skin tone of the patient, so long as thecolorant or opacifier does not interfere with the antiviral action ofthe formulation. Colorants include such compounds as, by way of exampleand without limitation, titanium dioxide, talc, FD&C Red No. 3, FD&C RedNo. 20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&COrange No. 5, FD&C Red No. 8, caramel, ferric oxide, other FD&C dyes,lakes, and natural coloring agents such as grape skin extract, beet redpowder, beta-carotene, annato, carmine, turmeric, paprika, and othermaterials known in the art. The amount of coloring agent used will varyas desired.

The topical composition carrier formulation should be stable andpharmaceutically acceptable. The composition should also enableincorporation of sufficient amounts of the active ingredients to givethe proper penetration characteristics. In addition to conventionalexcipient ingredients in lip-balms, sticks, creams, lotions, gels orointments, compositions based on phospholipids, including sphingolipidscan be advantageous. Various absorbent ointment bases, emulsion ointmentbases and water soluble ointment bases and components are known in theart and may be utilized in the compositions of the present disclosure,for example, as described in Remington's Pharmaceutical Sciences,Eighteenth Ed. 1990, Mack Publishing Co., Easton Pa., pp. 1311-1314.Ointment carrier bases may include, but are not limited to, waxes,petrolatum, esters of fatty alcohols, and saturated fatty acids, oleicacid, olive oil, paraffin, starch glycerin, lanolin, cetyl alcohol,glyceryl monostearate, methylparaben, propylparaben, glycol ethers,polyethylene glycols, polyoxyl 40 stearate, and polysorbates. Thecomposition may further comprise optional additional ingredientsselected from one or more of a penetration enhancer, oil, waxy compound,surfactant, stabilizer, gelling agent, moisturizer, water or apreservative.

Optional penetration enhancers serve to improve the absorption acrossthe skin of the antiviral compound. Penetration enhancers includevitamin E TPGS (Eastman Chemical Company, Kingsport, Tenn.), and othervitamin E derivatives as described in U.S. Pat. No. 6,193,985, which isincorporated herein by reference; and glyceryl monocaprylate/caprate(Cornwell et al. 1998, Int. J. Pharmaceutics, 171(2): 243-255). Otherpenetration enhancers are described in Smith and Maibach (eds.),Percutaneous Penetration Enhancers, CRC Press, Inc., Boca Raton, Fla.(1995), which surveys the use and testing of various skin penetrationenhancers, and Buyuktimkin et al., Chemical Means of Transdermal DrugPermeation Enhancement in Transdermal and Topical Drug Delivery Systems,Gosh T. K., Pfister W. R., Yum S. I. (Eds.), Interpharm Press Inc.,Buffalo Grove, I. L. (1997).

The oils, waxy compounds, gelling agents and surfactants selected forthe formulation and stabilization of these compositions are thosetraditionally employed in the dermatological arts. The optional oilsand/or waxy compounds can constitute from 0.5% to 99.9% of the totalweight of the composition. The amount of oil and/or wax depends on theactual form or physical state of the composition. Exemplary of such oilsare mineral oils (petrolatum); vegetable oils (sweet almond, macadamia,blackcurrant-pip oil); synthetic oils such as perhydrosqualene, fattyalcohols, acids or esters (octyl palmitate, isopropyl lanolate,triglycerides including those of capric/caprylic acids), oxyethylenatedor oxypropylenated fatty esters and ethers; and silicone oils(cyclomethicone, polydimethylsiloxanes or PDMS) or fluorinated oils.Exemplary waxy compounds include jojoba oil, paraffin, carnauba wax andbeeswax.

Exemplary surfactants (emulsifying and coemulsifying) include the estersof fatty acids and polyethylene glycol (PEG), esters of fatty acids andglycerol (glyceryl stearate) or esters of fatty acids and sugar(sorbitan stearate), as well as the polyoxyethylenated orpolyoxypropylenated derivatives thereof, cyclomethicones and dimethiconecopolyols, and also anionic surfactants (K or Na alkyl phosphate).

A preferred stabilizer includes glycol stearate or PEG-150 distearate.The stabilizer, when used, is typically present in an amount from about0.1 to 5 weight percent of the composition.

Exemplary gelling agents include modified clays (bentones), metal saltsof fatty acids (aluminum stearate), ethylene/acrylate copolymers,silicas, polyethylenes, calcium silicates or, alternatively, ethylcellulose.

Preferred moisturizers include wheat protein (e.g., laurdimoniumhydroxypropyl hydrolyzed wheat protein), hair keratin amino acids,sodium peroxylinecarbolic acid, panthenol, tocopherol (Vitamin E),dimethicone, and the like, and mixtures thereof. Sodium chloride mayalso be present, particularly when hair keratin amino acids are includedas a moisturizer. Moisturizers, when used, are typically present in anamount from about 0.01 to 2 weight percent, preferably about 0.05 to 1.5weight percent, more preferably from about 0.1 to 1 weight percent ofthe composition.

The water used is preferably deionized water.

Preferred preservatives include tetrasodium ethylene-diamine tetraaceticacid (EDTA), methylparaben, benzophenone-4, methylchloroisothiazolinone,methylisothiazolinone, and the like, and mixtures thereof Preservatives,when used, are typically present in an amount from about 0.01 to 6weight percent, preferably about 0.05 to 4 weight percent, and morepreferably from about 0.1 to 2 weight percent.

In one embodiment, the disclosure provides a composition comprising oneor more antiviral compounds and 2-deoxy-D-glucose in a lip-balm stickcarrier formulation. In one aspect, the lip balm stick carrierformulation is comprised of one or more polyethylene glycols (PEGs). Ina specific aspect, the lip-balm stick carrier formulation is comprisedof PEG 1450, PEG 300, silica gel, a flavoring and a sweetener. The finalmixture described above is poured, while still warm and fluid, intoappropriate tubes and allowed to cool until solid. The resulting lipbalm of the present disclosure is in the form of a stick. However, thelip balm of the present disclosure can also be marketed in a small widemouth jar. One preferred specific embodiment is shown in Example 4.

In another embodiment, the disclosure provides a composition comprisingacyclovir and 2-deoxy-D-glucose in an aqueous cream carrier formulation.Preparation of aqueous creams is described, for example, in U.S. Pat.No. 4,963,555, expired, which is incorporated herein by reference. Inone aspect, the aqueous cream comprises 5 wt % acyclovir and 0.2 wt %2-deoxy-D-glucose. A specific embodiment is shown in Example 5.

In another embodiment, the disclosure provides a composition comprisingacyclovir and 2-deoxy-D-glucose in an ointment carrier formulation. Inone aspect the ointment carrier formulation comprises a polyethyleneglycol.

In one embodiment, the disclosure provides a method of treating an HSVinfection outbreak by topically administering a composition comprisingan antiviral compound and 2-deoxy-D-glucose. A composition of thepresent disclosure can be administered topically to the affected area ina single daily dose or in multiple doses per day. In one aspect, thecomposition is administered four times a day. In another aspect, thecomposition is administered every three hours during waking hours. Inanother aspect, the composition is topically administered every twohours during waking hours. The treatment regimen may requireadministration from a single dose up to multiple daily doses for anextended period of time, for example, for several days or from one totwo weeks. In one specific aspect, the treatment regimen is topicalapplication of the composition every 2 hours during waking hours for 5days. The amount of antiviral compound and 2-deoxy-D-glucoseadministered per dose or the total amount administered will depend onsuch factors as the nature and severity of the infection, the age andgeneral health of the patient.

In one aspect, an equimolar combination of the two compounds acyclovirand 2-deoxy-D-glucose is unexpectedly more effective than eitherindividual compound at inhibiting HSV infection of primary Adult HumanDermal Microvascular Endothelial Cells in vitro. In vitro assays foranti-HSV-1 and anti-HSV-2 mRNA activity are described in Example 1.Confluent Vero cell monolayers were infected with HSV type 1 or HSV type2 at a multiplicity of infection of 10 and the resultant virus was usedto infect primary Adult Human Dermal Microvascular Endothelial Cells for1 hour; the supernatant was replaced with fresh culture media and thenincubated overnight in the presence of various concentrations ofacyclovir, 2-deoxy-D-glucose or an equimolar combination of the twocompounds. After overnight incubation, the cells were harvested, lysed,and subjected to QuantiGene Plex 2.0 assay (Panomics, Inc., Fremont,Calif.) for quantitative assay of HSV-1 and HSV-2 mRNA. The QuantiGenePlex 2.0 assay combines branched DNA (bDNA) signal amplification andmulti-analyte profiling beads (xMAP®) technologies to enable thedetection and quantitation of multiple mRNA targets simultaneously.

FIGS. 1 and 2 show concentration/response graphs for inhibition of HSV-1and HSV-2 infection of human endothelial cells when treated with variousconcentrations of acyclovir, 2-deoxy-D-glucose or an equimolarcombination of the two compounds. Particularly, FIG. 1 shows astatistically significant leftward shift of the dose-response curve forthe combination of acyclovir and 2-deoxy-D-glucose (IC₅₀=1.009 μM) ascompared to either acyclovir (IC₅₀=1.782 μM) or 2-deoxy-2-D-glucose(IC₅₀=20.9 μM). Thus, the combin of acyclovir and 2-deoxy-D-glucose isunexpectedly more effective than either individual compound atinhibiting HSV infection in vitro. The combination of acyclovir and2-deoxy-D-glucose can also be tested in vitro in either HSV-1 or HSV-2IgG ELISAs, as described in Examples 2 and 3.

In one embodiment, the disclosure provides a method of treating an HSVinfection outbreak by application of the composition of the presentdisclosure to a patient in need thereof. In one aspect, the disclosureprovides a method of decreasing the duration of an outbreak of herpeslabialis by application of the composition of the present disclosure toa patient in need thereof. In another aspect, application of thecomposition of the present disclosure reduces the recurrence ofoutbreaks of herpes labialis in a patient in need thereof. In oneaspect, the compositions of the present disclosure reduce the severityof outbreaks of herpes labialis in clinical patients. In another aspect,the compositions of the present disclosure reduce the pain associatedwith outbreaks of herpes labialis in clinical patients.

In a preliminary, non-placebo controlled clinical trial, patientsdiagnosed with herpes labialis were referred from a dentist with aprescription for acyclovir. Patients either had an ongoing outbreak, orwere known to be susceptible to recurrent outbreak following dentalwork. Patients received the lip-balm formulation of Example 4 asdescribed in Example 6. Patient surveys were collected. Data ispresented in Table 5. A reduction in the duration of the outbreak froman average of 10.8 days to 5.2 days was seen in patients who wereadministered the formulation of Example 4. The frequency in the numberof annual outbreaks was reduced from 6.2 to 1.9 per year. Generally, iftreatment was started early at prodrome, no visual outbreak would occur.A reduction in duration and severity of symptoms was seen if treatmentwas started at blister or early crust. Occasionally, outbreak persistedif treatment was started after prodrome, but an improvement in symptomsoccurred including relief of stinging and/or reduction in visualseverity. The formulation was considered pleasant.

EXAMPLES Example 1 In Vitro Assay for Anti-HSV-1 and Anti-HSV-2 mRNAActivity

Briefly, confluent Vero cell monolayers were infected with HSV type 1 orHSV type 2 at a multiplicity of infection of 10 and the resultant viruswas used to infect primary Adult Human Dermal Microvascular EndothelialCells for 1 hour and then the supernatant was replaced with freshculture media and then incubated overnight in the presence of variousconcentrations of acyclovir, 2-deoxy-D-glucose or an equimolarcombination of the two compounds. After overnight incubation, the cellswere harvested, lysed, and subjected to QuantiGene Plex 2.0 assay(Panomics, Inc., Fremont, Calif.) for quantitative assay of HSV-1 andHSV-2 mRNA. The QuantiGene Plex 2.0 assay combines branched DNA (bDNA)signal amplification and multi-analyte profiling beads (xMAP®)technologies to enable the detection and quantitation of multiple mRNAtargets simultaneously. Generally, this assay is considered to be a moresensitive and specific assay (bDNA) for the final readout when comparedto an ELISA. The ELISA method while easier, can lack specificity todetermine the difference between HSV1 and HSV2.

The bDNA assay is a hybridization-based method of target-specific RNAquantitation that amplifies signal rather than target RNA, using labeledDNA probes. The QuantiGene Plex 2.0 system utilizes fluorescentmicrospheres (Capture Beads) as a support to capture specific RNAmolecules. The ability to quantify multiple target-specific RNAmolecules in a single sample lies in the design of the Probe Sets. Foreach RNA molecule of interest, an oligonucleotide Probe Set containingthree types of synthetic probes, Capture Extenders (CEs), LabelExtenders (LEs), and Blockers (BLs) that hybridize and span contiguoussequences of the target RNA, is provided. The CEs discriminate among thedifferent Capture Beads within the bead array while capturing, viacooperative hybridization, the target RNA.

Signal amplification is mediated by DNA amplification molecules thathybridize to the tails of the LEs. Each amplification unit containsmultiple hybridization sites for biotinylated Label Probes that bindStreptavidin-conjugated R-Phycoerythrin (SAPE). The resultingfluorescence signal associated with individual Capture Beads is read ona Luminex flow cytometer. Signal is reported as median fluorescenceintensity (MFI) and is proportional to the number of target RNAmolecules present in the sample. All compounds were run in triplicatefor each concentration. Concentrations are expressed in micromolar (μM)concentrations.

The IC₅₀ (the drug concentration that reduces the number of virallyinfected cells by 50%) were determined by plotting the reduction influorescence (RLU) versus the drug concentration using Graph Pad Prismsoftware. Specifically, the variable slope model was used to calculatethe IC₅₀s with the formula“Y=Bottom+(Top−Bottom)/(1+10^((LogIC50−X)*HillSlope))” using Graph PadPrism software. The data is shown graphically in FIGS. 1 and 2. Theerror bars represent the standard error of the mean (SEM). Thestatistics for each evaluation are shown in Tables 1 and 2.

TABLE 1 IC₅₀ of Acyclovir and/or 2-Deoxy-D-glucose against HSV-1. 2Deoxy D Acyclovir + Nonlin fit of HSV1 Acyclovir glucose 2DDGlog(inhibitor) vs. Variable response -- slope Best-fit values LogIC500.2509 1.32 0.003803 HillSlope −1.108 −2.395 −1.406 IC50 1.782 20.91.009 Std. Error LogIC50 0.05128 0.03338 0.06276 HillSlope 0.1179 0.39510.2286 95% Confidence Intervals LogIC50 0.1463 to 1.252 to −0.1240 to0.3555 1.388 0.1316 HillSlope −1.349 to −3.200 to −1.871 to −0.8678−1.590 −0.9403 IC50 1.401 to 17.87 to 0.7517 to 2.267 24.45 1.354Goodness of Fit Degrees of Freedom 31 32 33 R² 0.9715 0.9377 0.9422Number of points Analyzed 34 35 36

TABLE 2 IC₅₀ of Acyclovir and/or 2-Deoxy-D-glucose against HSV-2. 2Deoxy D Acyclovir + Nonlin fit of HSV2 Acyclovir glucose 2DDGlog(inhibitor) vs. --Variable response slope Best-fit values LogIC500.2805 0.9029 0.212 HillSlope −1.113 −3.957 −1.543 IC50 1.908 7.9961.629 Std. Error LogIC50 0.05897 0.03075 0.05944 HillSlope 0.1338 0.83190.2717 95% Confidence Intervals LogIC50 0.1605 to 0.8402 to 0.09102 to0.4005 0.9655 0.3330 HillSlope −1.386 to −5.652 to −2.096 to −0.8410−2.262 −0.9895 IC50 1.447 to 6.922 to 1.233 to 2.515 9.237 2.153Goodness of Fit Degrees of Freedom 33 32 33 R² 0.9618 0.945 0.9366Number of points Analyzed 36 35 36

In these assays, the combination of the two compounds acyclovir and2-deoxy-D-glucose was unexpectedly more effective at inhibiting HSVinfection of cells in vitro than either compound individually, as shownby a comparison of the IC50 as well as the concentration/response graphsshown in FIGS. 1 and 2. Particularly, FIG. 1 shows a statisticallysignificant leftward shift of the dose-response curve for thecombination of acyclovir and 2-deoxy-D-glucose (IC₅₀=1.009 μM) ascompared to either acyclovir (IC₅₀=1.782 μM) or 2-deoxy-2-D-glucose(IC₅₀=20.9 μM).

Example 2 HSV-1 IgG ELISA

The presence of HSV IgG antibody is indicative of previous exposure. Asignificant increase in HSV IgG is indicative of reactivation, currentor recent infection. IgM antibody is present after primary HSVinfection. The CALBIOTECH INC. (CBI, Spring Valley, Calif., Cat. No.H1029G or H1029G4) HSV-1 ELISA Test system can be used to test for thepresence of HSV-1 IgG. A biological sample is subjected to the HSV-1ELISA which is an enzyme-linked immunosorbant assay for the detection ofIgG class antibodies to HSV-1 in human serum. Generally, the CBI HSV-1IgG ELISA package insert directions are followed. HSV-1 antigen coatedwells are provided, along with positive and negative controls andcalibrators. For example, diluted patient serum is added to wells coatedwith purified antigen. IgG specific antibody, if present, binds to theantigen. All unbound materials are washed away and the enzyme conjugateis added to bind to the antibody-antigen complex, if present. Excessenzyme conjugate is washed off and TMB substrate is added. The plate isincubated at room temperature for 10 minutes to allow the hydrolysis ofthe substrate by the enzyme. Stop solution is added after 10 minutes.The ELISA plate O.D. is read at 450 nm. The intensity of the colorgenerated is proportional to the amount of IgG specific antibody in thesample. Generally, an antibody index <0.9 is interpreted as nodetectable antibody to HSV-1 IgG by ELISA; an antibody index of 0.9-1.1is a borderline positive; and an antibody index >1.1 indicatesdetectable antibody to HSV-1 IgG by ELISA.

Example 3 HSV-2 IgG ELISA

The presence of HSV IgG antibody is indicative of previous exposure. Asignificant increase in HSV IgG is indicative of reactivation, currentor recent infection. IgM antibody is present after primary HSVinfection. The CALBIOTECH INC. (CBI, Spring Valley, Calif., Cat. No.H2031G or H2031G4) HSV-2 ELISA Test system can be used to test for thepresence of HSV-2 IgG. A biological sample is subjected to the HSV-2ELISA which is an enzyme-linked immunosorbant assay for the detection ofIgG class antibodies to HSV-2 in human serum or plasma.

Generally, the CBI HSV-2 IgG ELISA package insert directions arefollowed. HSV-2 antigen coated wells are provided, along with positiveand negative controls and calibrators. For example, diluted patientserum is added to wells coated with purified antigen. IgG specificantibody, if present, binds to the antigen. All unbound materials arewashed away and the enzyme conjugate is added to bind to theantibody-antigen complex, if present. Excess enzyme conjugate is washedoff and TMB substrate is added. The plate is incubated at roomtemperature for 10 minutes to allow the hydrolysis of the substrate bythe enzyme. Stop solution is added after 10 minutes. The ELISA plateO.D. is read at 450 nm. The intensity of the color generated isproportional to the amount of IgG specific antibody in the sample.Generally, an antibody index <0.9 is interpreted as no detectableantibody to HSV-2 IgG by ELISA; an antibody index of 0.9-1.1 is aborderline positive; and an antibody index >1.1 indicates detectableantibody to HSV-2 IgG by ELISA.

Example 4 Antiviral Topical Formulations. Lip-Balm Formulation

Ingredients for one batch of a lip-balm formulation comprising about 5%w/w of acyclovir and 0.2% w/w 2-deoxy-D-glucose are provided in Table 3.

TABLE 3 Lip-balm Formulation. specific Amount per % Ingredient Amountgravity chapstick (w/w) Acyclovir, USP 1.6667 g 0.333 g 4.52-Deoxy-D-glucose 0.0667 g 0.0133 g 0.2 Silica gel, USP/NF powder 0.200g 0.04 g 0.5 Stevioside, 90% powder 0.3333 g 0.0667 g 0.9 Polyethyleneglycol 1450, 16.25 g 3.25 g 43.6 NF granules Polyethylene glycol 300,16.6667 mL 1.1 3.33 mL 49.1 NF liquid Flavor, spearmint oil 0.5 mL 0.9170.1 mL 1.2

The polyethylene 1450 and 300 was melted at 50° C. with stirring. Theacyclovir, 2-deoxy-D-glucose, silica gel and stevioside were trituratedtogether. The triturated powders were slowly sifted into the melted PEGswith stirring. The flavoring was added, followed by thorough mixing. Themixture was poured into applicator tubes and allowed to cool to roomtemperature. Amounts of acyclovir and 2-deoxy-D-glucose employed may bevaried in accordance with the specification.

Example 5 Antiviral Topical Formulations. Aqueous Cream Formulation

Ingredients for one batch of an aqueous cream formulation comprisingabout 5% w/w of acyclovir and 0.2% w/w 2-deoxy-D-glucose are provided inTable 4.

TABLE 4 Aqueous Cream Formulation. Ingredient Amount % (w/w) Acyclovir,USP 50.0 g 5 2-Deoxy-D-glucose 2.0 g 0.2 Cetostearyl alcohol 67.5 gSodium lauryl sulphate 7.5 g White soft paraffin 125.0 g Liquid paraffin50.0 g Propylene glycol 400.0 g Purified water, Q.S. to 1000.0 g

A part of the acyclovir (5 g) is dissolved in the water with the2-deoxy-D-glucose and propylene glycol at ambient temperature to producean aqueous solution. The paraffins and emulsifiers (cetostearyl alcoholand sodium lauryl sulphate) are mixed together and heated to 60° C., andemulsified with the aqueous solution, also at 60° C., using a laboratorymixer. The remaining acyclovir is added, the mixture dispersed, allowedto cool, and filled into lacquered aluminum tubes. Amounts of acyclovirand 2-deoxy-D-glucose employed may be varied in accordance with thespecification.

Example 6 Clinical Results

Patients diagnosed with herpes labialis were referred from a dentistwith a prescription for acyclovir. Patients had an ongoing outbreak, orwere known to be susceptible to recurrent outbreak following dentalwork. A patient population comprising about 40 patients received thelip-balm formulation of Example 4. Data to verify treatment outcomes,adverse effects and patient comment was collected by patient surveyseveral months after dispensing the formulation. Sixteen patientsreturned the survey to date. Surveyed patients ranged in age from 14 to65 years old, with an average age of 42 years old. Nine males and sevenfemales returned the survey. Patients used the formulation for anaverage of 4.8 days on average every 2-3 hours following outbreak signsof prodrome or early crust. Table 5 shows data collected from patientsurveys.

TABLE 5 Clinical Data. Duration of Frequency of outbreak outbreakPatient Gender Days Number per year No. Age F M Prior During use PriorDuring use 1 28 1 0 10 5 12 6 2 14 0 1 12 6 7 2.5 3 44 1 0 10.5 3 6 1 453 1 0 14 7 2 1 5 65 0 1 7 5 4.5 0 6 38 0 1 14 9 5 3 7 39 1 0 14 7 2.51.5 8 62 0 1 4 2 4 0 9 40 1 0 7 1.5 4 n/a 10 55 0 1 10.5 5 17 1 11 42 10 12 6 6 n/a 12 38 0 1 10 6 5 n/a 13 17 0 1 12 8 2.5 n/a 14 48 0 1 11 44.5 n/a 15 45 0 1 12 2 4.5 n/a 16 45 1 0 12 7 13 3 Average 42.1 7.0 9.010.8 5.2 6.2 1.9 Std. Dev. 14.0 2.8 2.2 4.2 1.9

A reduction in the duration of the outbreak from an average of 10.8 daysto 5.2 days was seen in all patients surveyed. The frequency in thenumber of annual outbreaks was reduced from 6.2 to 1.9 per year.Generally, if treatment was started early at prodrome, no visualoutbreak would occur. A reduction in duration and severity of symptomswas seen if treatment was started at blister or early crust.Occasionally outbreak persisted if treatment was started after prodrome,but an improvement in symptoms occurred; including relief of stinging,reduction in visual severity and/or shortened crusting time. Patientacceptability was good with comments on pleasant smell.

Example 7 Further Clinical Results

As in Example 5, patients diagnosed with herpes labialis were referredfrom a dentist with a prescription for acyclovir. Patients had anongoing outbreak, or were known to be susceptible to recurrent outbreakfollowing dental work. Most patients had previously been treated withone or more of Zovirax® (topical or oral acyclovir), Valtrex® (oralvalacyclovir hydrochloride), Famvir® (oral famciclovir), and/or over thecounter medications Abreva® (topical 10% docosanol), Zilactin® (topical10% benzyl alcohol), Novitra® (topical zincum oxydatum 2X) andCampho-Phenique® (topical camphor 10.8% and phenol 4.7%). The patientpopulation received the lip-balm formulation of Example 4. Data toverify treatment outcomes, adverse effects and patient comment wascollected by patient survey several months after dispensing theformulation.

One patient population started application of the topical testformulation at a prodrome stage comprising tingling and/or pain, butprior to outbreak. The data for patients who started treatment bytopical application of the test formulation at prodrome, prior to fulloutbreak, is shown in Table 6.

TABLE 6 Clinical Data for Treatment Started at Prodrome. ProductApplication Duration of Average outbreak Days Patient Gender Days DuringNo. Age F M Applied Times/Day Prior Use 1 22 0 1 15.5 5.5 10.5 14 2 48 01 9 3.5 4 9 3 51 1 0 1.5 3.5 2 2 4 24 1 0 7.5 5.5 7 6 5 38 0 1 5.5 3.5 75 6 50 0 1 7.5 5.5 10 6.5 7 56 1 0 11.5 5.5 14 12 8 20 1 0 7.5 3.5 6 6 944 0 1 5.5 3.5 6 1.5 10 53 0 1 3.5 3.5 5 3 11 49 1 0 3.5 3.5 5 1 12 31 10 3.5 5.5 7 4 13 36 1 0 1.5 1.5 9 0.5 14 33 0 1 3.5 5.5 10 4 15 45 1 01.5 3.5 8 2 16 45 1 0 7.5 5.5 6.5 3 17 52 0 1 3.5 3.5 10 4 18 45 0 1 5.55.5 12 2 19 48 0 1 5.5 5.5 4.5 4 20 41 0 1 1.5 1.5 5 3 21 22 0 1 11.55.5 14 6 22 59 1 0 3.5 1.5 6 2 23 67 1 0 3.5 3.5 3 3 24 63 1 0 3.5 5.5 73 Average 43.4 12.0 12.0 5.6 4.2 7.4 4.4

When topical treatment with the formulation of Example 4 was started atprodrome, the average reduction in duration of outbreak was about threedays. Twenty-one of the 24 patients first treated at prodrome preferredthe test formulation to other medications. All six of the six patientswho reported previous use of Valtrex®, preferred the test formulation.Four of four patients who reported previous use of Zovirax® preferredthe test formulation. Patients also generally preferred the testformulation to Abreva® (18), Zilactin® (3), Novitra® (2), andCamphophenique® (1).

Alternatively, another patient population started the test formulationat outbreak; or at first blister, weeping or crusting stage. The datafor patients who started treatment by topical application of the testformulation of Example 4 after outbreak is shown in Table 7.

TABLE 7 Clinical Data for Treatment Started after Outbreak Duration ofProduct outbreak Application Days Patient Gender Days During No. Age F MApplied Times/Day Prior use 1 38 0 1 5.5 3.5 7 5 2 55 1 0 7.5 5.5 7 5 331 0 1 7.5 3.5 6 7 4 33 1 0 3.5 5.5 7 4 5 13 0 1 3.5 7.5 6 4 6 34 0 13.5 5.5 10 6 7 49 1 0 3.5 5.5 6 3 8 31 0 1 3.5 5.5 6 5 9 34 1 0 5.5 5.514 6.5 10 25 0 1 7.5 5.5 7 7 11 38 1 0 3.5 5.5 6 3 12 45 0 1 5.5 5.5 127 13 54 1 0 9.5 3.5 10 8 14 33 1 0 5.5 5.5 5 2 15 17 0 1 3.5 5.5 12 8 1638 1 0 3.5 5.5 8.5 3.5 17 18 1 0 5.5 5.5 14 7 18 32 1 0 5.5 5.5 5 3 1937 1 0 1.5 5.5 11 3 20 9 1 0 3.5 3.5 7 6 21 50 0 1 5.5 1.5 11 6 22 33 10 5.5 3.5 6 4 23 53 0 1 5.5 3.5 14 6 24 59 0 1 7.5 3.5 5 3 Average 35.813.0 11.0 5.1 4.8 8.4 5.1

When topical treatment with the formulation of Example 4 was startedafter outbreak, the average reduction in duration of outbreak was about3.3 days. However, two of 24 patients treated reported no benefit.Twenty of the 24 patients first treated after outbreak preferred thetest formulation to other medications. Of four patients that hadpreviously used Valtrex®, three preferred the test formulation. Of twopatients that had previously used Zovirax®, one preferred the testformulation. Patients also generally preferred the test formulation toAbreva® (14), Zilactin® (4), Novitra® (4), and Camphophenique® (2).

In general, regardless of whether the topical test medication wasstarted at prodrome or after outbreak, patients made multiple commentsthat the blisters disappeared, ulcer crusting time appeared to beshortened, and the tingling and pain stopped. In addition, very littleto no scabbing occurred.

Example 8 Clinical Survey in HSV-2 Patient

A 23 year old female diagnosed with HSV-2 typically experienced aboutone recurrent outbreak per year with prodrome symptoms of tingling,soreness, and tenderness around the leg. Outbreak triggers includedstress, sunlight, fatigue and hormonal changes. Typical duration of anoutbreak was about 7 days. At the first signs of outbreak, includingtingling and pain, the patient treated the affected area 3 to 4 times aday for seven days with a topical stick test formulation comprising 4.5wt % acyclovir and 0.2 wt % 2-deoxy-D-glucose. The formulation employedwas similar to that of Table 3, but without sweetener or flavoring. Thepatient experienced cessation of tingling and pain within 2 days and nofull outbreak occurred. The patient reported that the test medicationsuppressed the HSV outbreak better than Valtrex® when administered twicea day.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the disclosure.Those skilled in the art will readily recognize various modificationsand changes that may be made to the present disclosure without followingthe example embodiments and applications illustrated and describedherein, and without departing from the true spirit and scope of thepresent disclosure, which is set forth in the following claims.

1. A topical pharmaceutical composition comprising a therapeuticallyeffective amount of antiviral compound acyclovir; and a therapeuticallyeffective amount of 2-deoxy-D-glucose; wherein the composition comprisesa molar ratio of acyclovir to 2-deoxy-D-glucose in a range from about1:1 to about 18.2:1; and wherein the composition comprises acyclovir ina wt % range of from about 3 wt % to about 5 wt %, and 2-deoxy-D-glucosein a wt % range of about 0.1 wt % to about 0.2 wt %.
 2. The compositionof claim 1 in a form selected from a lip-balm, stick, cream or ointment.3. The composition of claim 2 in a lip-balm form.
 4. The composition ofclaim 3, further comprising one or more polyethylene glycols.
 5. Thecomposition of claim 4, further comprising one or more sweeteners orflavorings.
 6. The composition of claim 5 wherein the sweetener isstevioside.
 7. The composition of claim 5 wherein the flavoring isspearmint oil.
 8. The composition of claim 1 wherein the topicalcomposition comprises about 5 wt % of acyclovir and about 0.2 wt % of2-deoxy-D-glucose.
 9. A method of treating an HSV-1 herpes viralinfection of the skin or mucosa of a patient in need thereof comprisingtopically administering to the skin or mucosa a pharmaceuticalcomposition comprising a therapeutically effective amount of antiviralcompound acyclovir, and a therapeutically effective amount of2-deoxy-D-glucose; wherein the composition comprises a molar ratio ofantiviral compound to 2-deoxy-D-glucose in a range from about 1:1 toabout 18.2:1; and wherein the composition comprises acyclovir in a wt %range of from about 3 wt % to about 5 wt %, and 2-deoxy-D-glucose in awt % range of about 0.1 wt % to about 0.2 wt %.
 10. The method of claim9, wherein the topical composition is in a form selected from alip-balm, stick, cream or ointment.
 11. The method of claim 10, whereinthe topical composition is in a lip-balm form.
 12. The method of claim11, wherein the topical composition further comprises one or morepolyethylene glycols.
 13. The method of claim 11, wherein the topicalcomposition further comprises one or more sweeteners or flavorings. 14.The method of claim 13, wherein the sweetener is stevioside.
 15. Themethod of claim 13, wherein the flavoring is spearmint oil.
 16. Themethod of claim 9, wherein the topical composition comprises about 5 wt% of acyclovir and about 0.2 wt % of 2-deoxy-D-glucose.
 17. The methodof claim 9 wherein the treatment is for latent infection of herpessimplex type 1 infection.
 18. A method of reducing the duration of anHSV-1 herpes viral infection outbreak of the skin or mucosa of a mammalin need thereof comprising administering to the skin or mucosa apharmaceutical composition comprising a therapeutically effective amountof antiviral compound acyclovir, and a therapeutically effective amountof 2-deoxy-D-glucose; wherein the composition comprises a molar ratio ofantiviral compound to 2-deoxy-D-glucose in a range from about 1:1 toabout 18.2:1; and wherein the composition comprises acyclovir in a wt %range of from about 3 wt % to about 5 wt %, and 2-deoxy-D-glucose in awt % range of about 0.1 wt % to about 0.2 wt %.
 19. The method of claim18, wherein the antiviral compound is acyclovir.
 20. The method of claim18 wherein the treatment is for latent infection of herpes simplex type1 infection.
 21. The method of claim 17 wherein the infection is anorofacial herpes infection.
 22. The method of claim 18 wherein theinfection is an orofacial herpes infection.
 23. The method of claim 20wherein the infection is an orofacial herpes infection.
 24. The methodof claim 18 wherein the topical composition comprises about 5 wt % ofacyclovir and about 0.2 wt % of 2-deoxy-D-glucose.